Neurons are the most prolific users of calcium signaling, yet many of the pathways that lead to cell death require calcium. This would appear to place neurons at special risk for degeneration as a result of failed calcium homeostasis. The isolation and characterization of an unusual class of calmodulin-binding peptide may highlight one mechanism for how neurons cope with frequent and sometimes vigorous calcium influx. This class of peptide binds the inactive form of calmodulin and inhibits its conversion to a form that can activate biological targets. Known members of this class are the neuronal peptides GAP-43 and neurogranin, and PEP-19 is likely to become a new member. Consistent with its abundant use of calcium-mediated signaling, the brain is also the richest source of the calcium-signal transducer, calmodulin and calmodulin inhibition has been shown to be beneficial in slowing cell death. It therefore appeared likely that GAP-43, neurogranin and PEP-19 could function as endogenous calmodulin inhibitors with a concomitant function of protecting neurons from degenerative conditions brought on by unbridled metabolic stimulation. The first project will expand the enzyme inhibition studies for GAP-43 in order to determine if its effect on calmodulin-dependent enzyme activation is a general one. This will be accomplished by examining GAP-43 action on calmodulin-dependent adenylate cyclase. The next two sets of experiments will study the ability of GAP-43 isoforms to differentially regulate calmodulin and examine if native PEP-19 has calmodulin regulatory properties. Both projects will use nitric oxide synthase as the prototypical calmodulin target. Because it appears that the brain contains several other peptides which may function like GAP-43 or neurogranin, the next project will characterize a major peptide from brain that had been isolated on calmodulin affinity chromatography. This peptide is related to ARPP 19 and may be another calmodulin regulator with different features of control. The final project will exploit what we have learned about these peptides from in vitro studies and begin to test them for metabolic consequences in cell culture. This will be examined by following the susceptibility of naive PC12 cells to death as a function of calcium- dependent challenge.